Flagellin is the generic name for the main structural protein that makes up bacterial flagella. They are cylindrical structures of variable length (approx. 530 nm) and about 21 nm in diameter [1]. The flagella are observed in both Gram-positive and Gram-negative bacteria; they are structures of variable length that allow bacteria to move in liquid media. In addition to being formed by flagellin, the bacterial flagellum is also composed of many other proteins that intervene in the assembly, the interaction with the cell's external envelopes, or participate in chemotactic processes.
The X-ray diffraction study of the structure of bacterial flagellin from the genus Salmonella has made it possible to increase the knowledge about its function and biological implications [1, 2]. Flagellin may play a significant role in bacterial pathogenesis [3] and has been defined as a prototype of “pathogen-associated molecular patterns” (PAMPs), since it is capable of activating the innate immune system through interaction with specific receptors [4], [5].
Most bacterial flagellins are recognised by the “Toll-like-5” (TLR5) receptor, which is located in the membrane of epithelial cells and immune system cells: monocytes, T lymphocytes, NK cells and immature dendritic cells. TLR5 is one of the receptors in the “Toll-like” family which have the capacity to interact with PAMPs [6], each TLR having the capacity to recognise specific PAMPs [7]. Once the flagellin is bound to TLR5, a signal transduction cascade is initiated through MyD88 (Myeloid differentiation primary response gene 88) in order to mediate in the production of cytokines necessary for the development and regulation of an innate and adaptive immune response in the host [8-10]. The binding of Salmonella flagellin to TLR5 is very specific and acts with a high affinity, at concentrations as low as 8.5×10−10 M [11]. On the other hand, it is also well-known that some bacterial flagellins are not capable of activating the immune system when a natural infection occurs, that is, they do not bind to the TLR5 receptor to induce the inflammatory response [12]. The immunological escape of these flagellins has been circumscribed to bacteria from the alpha and epsilon subgroups (Helicobacter pylori, for example), whereas responding flagellins, those which activate the host's immune system, would belong to the beta and gamma groups [12]. The molecular and functional analysis of non-responding flagellins, those which do not activate the host's immune system, made it possible to define a specific interaction region with the TLR5 receptor in flagellins, this specific region being that comprised between amino acids 89-96 of the flagellin protein sequences [13].
Most current vaccines are composed of the antigen of interest and adjuvants [14], [15]. Although adjuvants improve the immune response, they may also cause adverse secondary effects, as in the case of Freund's complete adjuvant [16], [17], or even in the case of other adjuvants approved by the FDA or the EMEA. In order to solve said problem associated with the manufacturing of vaccines and improve the effectiveness thereof, the Salmonella typhimurium flagellin has been used as a vaccine adjuvant, since it has been shown that this flagellin, in transcriptional fusion with peptides or proteins, induces a humoural and cellular immune response, innate and adaptive, toward them, which is very rapid and potent. Some of the vaccines based on the Salmonella flagellin have been aimed against cholera [18], influenza [19], 20[20], plague (Yersinia pestis) [21], 22[22], malaria (Plasmodium falciparum) [23] and the West Nile virus [24].
The Salmonella bacterium is currently classified into the species S. bongori and S. enterica [25]. Most Salmonella that infect mammals and birds belong to S. enterica, which is divided into six subspecies (enterica, salamae, arizonae, diarizonae, houtenae, indica), with approximately 2,000 serotypes defined on the basis of differences in the composition of lipopolysaccharides (LPS) and flagellar antigens [25]. Some serotypes are host-exclusive, such as S. typhi (humans) and S. pullorum (birds), and others are primarily adapted to specific hosts, such as S. cholerae-suis (porcines), S. abortus-ovis (ovines), and S. dublin (bovines) [25].
Infection by Salmonella causes the appearance of cellular and humoural immunity against various antigens of said bacteria. One of these antigens is flagellin, as has been shown in various studies performed on people of Caucasian origin in Denmark and the United States [26]-[27]. In a random population study in the U.S., 30% of the subjects had antibodies against the Salmonella flagellin [28]. On the other hand, the response capacity toward flagellin in bacterial infections is diverse, and high responders and low responders to re-immunisation with flagellin may be found [27]; these results are attributed to certain Gm genotypes of flagellin [27]. Likewise, birds and other animals are very susceptible to infection by enteric Salmonella [29]. Therefore, infections by Salmonella occur in different species and countries, which indicates the scope of these pathologies; for this reason, control programmes for humans have been implemented for many years, and it is a mandatory declaration disease in bovines, ovines and caprines. All these data indicate that a significant percentage of the population that is infected presents antibodies against the Salmonella flagellin.
Most variants of Salmonella have two types of different genes that encode flagellin, although only the flagellin from one of these genes is expressed at each time. The bacterium is capable of alternating the expression of the two flagellins, called phase-1 flagellin and phase-2 flagellin. The operon that controls the synthesis of phase-1 flagellin also encodes a repressor of the synthesis of phase-2 flagellin, and vice-versa. The change mechanism from phase 1 to phase 2 in the synthesis of flagellin may be a consequence of the bacteria's attempt to avoid cellular immunity.
A recent study [30] has provided much clarification about those aspects related to the immunogenicity of the Salmonella typhimurium flagellin. Mice that are immunised with the flagellin on successive occasions produce antibodies that neutralise the TLR5-flagellin interaction capacity. These antibodies are primarily aimed at the hypervariable region (HPVR) of the flagellin. The obtainment of various flagellins the hypervariable regions whereof are eliminated to different extents has made it possible to observe that these modified flagellins preserve their capacity to interact with TLR5, and react to a lesser extent with a hyperimmune mouse serum obtained from immunisation with wild-type flagellin. However, the reactivity of a hyperimmune mouse serum following immunisation with a version of flagellin without HPVR between the wild-type flagellin and others without HPVR is very similar, which indicates the presence of antibodies that neutralise the conserved regions of the flagellins, including the regions of interaction with TLR5. On the other hand, the complete elimination of the HPVR prevents the modified flagellin from normally initiating the production of inflammatory cytokines (CCL20 and CXCL2) at the systemic level, although this alteration in the flagellin does not cause the same effect at the mucous membrane level [30]. That is, when this flagellin is injected, it fails to produce inflammatory mediators, but it has an adequate behaviour when it is applied to mucous membranes. In summary, all these data indicate that:
1. The function of flagellin may be neutralised by pre-existing antibodies against the wild-type flagellin (complete). The antibodies generated against a wild-type flagellin are primarily directed against the hypervariable region.
2. If the hypervariable region is completely eliminated, the anti-flagellin antibodies generated recognise the conserved domains, which include the region of interaction with TLR5. Therefore, it would be preferable not to eliminate the hypervariable region, since it is more immunodominant and, thus, the antibodies generated are not directed against the region of interaction with TLR5.
3. Different versions of flagellin without HPVR fragments are functional upon activating TLR5 at the mucous membrane level, but do not work equally well at the systemic level, except for one of the versions obtained, which has an HPVR part, and does behave like the wild-type flagellin. However, it may be neutralised, albeit to a lesser extent than the wild type, by serum antibodies that have reacted against the wild-type flagellin [30].
All of the above suggests that a vaccine based on the Salmonella flagellin capable of activating the immune system in all its forms could not be used repeatedly because the pre-existing antibodies would neutralise the potential thereof.
For all these reasons, a very significant advance would be made in the development and application of flagellin-based vaccines if other flagellins were available that induce the same activation of the innate immune system and escape the immunological response triggered against the Salmonella flagellins.
In this regard, this invention has surprisingly discovered that vaccines based on flagellins from marine bacteria of the species Marinobacter algicola are equally effective, or even more so, than vaccines based on Salmonella flagellins. Since Marinobacter algicola bacteria are marine bacteria, prior infection of non-aquatic organisms is very unlikely. To this date, it has not even been determined whether said bacteria are capable of infecting aquatic organisms. Thus far, 12 species of Marinobacter have been recognised, isolated from different sources, such as crude oil extraction ducts in off-shore platforms [31], coastal thermal waters [32], hot springs [33] and Antarctic waters [34], saline soils [35] and marine sediments [36]. In laboratory studies of different algae, three bacterial strains from the genus Marinobacter have been isolated, which have been called Marinobacter algicola, two of them originating in the Yellow Sea off the coast of Korea (DG893T and DG1136) and one from the Vigo Estuary in Galicia (GC21V) [37].
As in the case of Salmonella, the Marinobacter algicola bacterium has two genes that encode two flagellins. Thus, this invention describes the use of the two recombinant flagellin protein sequences from the species Marinobacter algicola (DG893T strain), called flagellin F and flagellin FR, capable of inducing and developing a specific immune response by themselves, without the aid of other adjuvants, which indicates that said flagellins are capable of appropriately interacting with the TLR5 receptor, triggering the immunological response. But this invention also shows that Marinobacter algicola flagellins F and FR may fuse to peptides, proteins or vaccinal antigens, or be jointly administered with said peptides and proteins, without being fused thereto, and developing a specific immune response.
Moreover, it shows the limited cross-reactivity between the flagellins from the genus Marinobacter described in this invention and the Salmonella flagellin. This limited reactivity between flagellins from both species is due to the relatively low sequence homology between both flagellins (33%-36%). It is worth noting that the low cross-reactivity between said flagellins represents an advantage in the effectiveness of vaccines based on Marinobacter flagellins, since, as previously discussed, there could be partial neutralisation of vaccines based on the Salmonella flagellin in subjects that have been infected by or had previous contact with Salmonella, or have been previously immunised with the Salmonella flagellin. However, prior immunity against bacteria from the genus Marinobacter is unlikely, since it is a marine bacterium that is not known to infect human beings or other organisms, and the likelihood of contact with non-marine organisms to produce an immunological response therein is very low.
We also describe different combined vaccination strategies based on the use of the two Marinobacter flagellins described in this invention and the Salmonella typhimurium flagellin. In this regard, subjects that have had prior contact with Salmonella could be vaccinated using the Marinobacter flagellins (F and FR), whereas in subjects not previously exposed to Salmonella a different strategy could be used: in the first place, they could be immunised with a vaccine based on the Marinobacter flagellin and, subsequently, another vaccine based on the Salmonella flagellin could be applied. This would enhance the use of these vaccines for multiple antigens without reducing the efficacy thereof due to cross-seroneutralisation between the flagellins.